Process for the production of prenyl diphosphate using mutants of geranylgeranyl diphosphate synthase

ABSTRACT

The present invention discloses a mutated enzyme comprising a geranylgeranil diphosphate synthase having its origin in wild type Sulfolobus acidocaldarius wherein at least one of phenylalanine at position 77, methionine at position 85, valine at position 99, tyrosine at position 101, phenylalanine at position 118, arginine at position 199 and aspartic acid at position 312 is substituted with another amino acid.

This application is a division of allowed application Ser. No.08/705,377 filed Aug. 29, 1996 now U.S. Pat. No. 5,807,725, the entiretyof which is incorporated herein by reference.

BACKGROUND OF INVENTION

1. Field of Invention

The present invention relates to a mutant prenyl diphosphate synthasethat is able to synthesize prenyl diphosphate having a longer chain thanprenyl diphosphate synthesized by the native prenyl diphosphatesynthase.

2. Related Art

Prenyl diphosphate is highly valuable in biosynthesis pathways,functioning as a precursor of steroids, a precursor of carotenoids,being a transition substrate of prenylated proteins, being a substratefor synthesis of vitamin E, vitamin K and ubiquinone (CoQ) and so forth.Prenyl diphosphate exists in various forms, including dimethylallyldiphosphate (DMAPP; C5), geranyl diphosphate (GPP; C10), arnesyldiphosphate (FPP; C15), geranylgeranyl diphosphate (GGPP; C20),geranylfarnesyl diphosphate (GFPP; C25), hexaprenyl diphosphate (HPP;C30), heptaprenyl diphosphate (HepPP; C35) and octaprenyl diphosphate(OPP; C40).

Prenyl transferases, which synthesize these prenyl diphosphates, areenzymes that form prenyl diphosphate by continuously condensingisopentenyl diphosphate (IPP; C5) into allylic diphosphate, and exist invarious forms, including farnesyl diphosphate synthase (FPS),geranylgeranyl diphosphate synthase (GGPS), geranylfarnesyl diphosphatesynthase (GFPS), hexaprenyl diphosphate synthase (HexPS), heptaprenyldiphosphate synthase (HepPS) and octaprenyl diphosphate synthase (OPS).

However, among the above-mentioned prenyl diphosphates, only those fromdimethylallyl diphosphate having 5 carbon atoms to geranyl diphosphatehaving 20 carbon atoms are commercially available in small amounts asreagents, and a process for industrially synthesizing and recoveringlarge amounts of prenyl diphosphates having longer chains is not known

The carbon chain length and stereoisomerism of synthesized prenyldiphosphates are known to be specifically determined depending on theparticular enzyme. Until now, it has not been clear what type ofmechanism is the factor in determining carbon chain length.

Although prenyl transferases and their genes are known to be derivedfrom bacteria, mold, plants and animals, these enzyme are typicallyunstable, difficult to handle and are not expected to be industriallyvaluable.

The prenyl transferases and their genes of thermophilic organisms, whichare stable and easy to use as enzymes, are only farnesyl diphosphatesynthase (FPS) (Koyama, T. et al. (1995) J. Biol. Chem. 113, 355-363)and heptaprenyl diphosphate synthase (HepPS) (Koike-Takeshita, A. et al.(1995) J. Biol. Chem. 270, 18396-18400) from the moderately thermophilicarchaebacterium, Bacillus stearothermophilus; geranylgeranyl diphosphatesynthase (GGPS) from the hyper thermophilic bacterium, Sulfolobusacidocaldarius (Ohnuma, S.-i. et al. (1994) J. Biol. Chem. 268,14792-14797); as well as farnesyl diphosphate/geranylgeranyl diphosphatesynthase (FPS/GGPS) from the methane-producing archaebacterium,Methanobacterium thermoautotrophicum (Chen, A. and Poulter, C. D. (1993)J. Biol. Chem. 268, 11002-11007). Only HepPS can synthesize prenyldiphosphate having 35 carbon atoms, and enzymes having thermal stabilitythat synthesize prenyl diphosphates having 25 or more carbon atoms havenot been reported. In addition, the above-mentioned HepPS does not haveadequate heat resistance, is composed of two types of subunits, andhandling is not always easy.

SUMMARY OF INVENTION

Thus, the present invention provides a thermostable prenyl diphosphatesynthase capable of synthesizing long-chain prenyl diphosphate, aprocess for its production, and a method for using said enzyme.

In order to create an enzyme that can synthesize prenyl diphosphatehaving a longer chain length, the inventors of the present inventionsucceeded in creating a mutant enzyme able to synthesize prenyldiphosphate having a longer chain than naturally-occurringgeranylgeranyl diphosphate synthase by treating DNA coding forgeranylgeranyl diphosphate synthase with a mutation agent, introducingthe above-mentioned treated DNA into the yeast, Saccharomycescerevisiae, deficient for hexaprenyl diphosphate synthase activity, andselecting a mutant DNA that can complement the above-mentioneddeficient, and moreover, elucidated the relationship between themutation site in the enzyme and the chain length of the prenyldiphosphate that is formed, thereby leading to completion of the presentinvention.

Thus, the present invention provides a mutant enzyme wherein, least oneof phenylalanine residue at position 77, methionine residue at position85, valine residue at position 99, tyrosine residue at position 101,phenylalanine residue at position 118, Arginine residue at position 199and aspartic acid residue at position 312 in a geranylgeranyldiphosphate synthase of Sulfolobus acidocaldarius origin is substitutedwith another amino acid, and which enzyme can synthesize prenyldiphosphate having at least 25 carbon atoms.

Moreover, the present invention provides a gene system that codes forthe above-mentioned enzyme, and a process for producing theabove-mentioned enzyme using that gene system.

Furthermore, the present invention provides a process for producing amutant prenyl diphosphate synthase comprising the steps of culturing ahost transformed with a gene in which the codon for phenylalanineresidue located at the fifth N-terminal side position from theN-terminal amino acid of the aspartate-rich domain I in a gene thatcodes for the native enzyme, is converted to a codon for a non-aromaticamino acid, thereby enabling the expression of a mutant enzyme that isable to synthesize prenyl diphosphates having a longer chain than thelongest chain of prenyl diphosphate synthesized by the native prenyldiphosphate synthase.

In addition, the present invention provides a process for producinglong-chain prenyl diphosphate using the above-mentioned enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 indicates the mutation site of the present invention in thegeranyl diphosphate synthase derived from Sulfolobus acidocaldarius. Thearrows in the drawing indicate two aspartate-rich domains.

FIG. 2 is photograph that indicates the autoradiograph of a thin layerchromatography which shows the products in the case of allowing themutant enzymes of the present invention produced in yeast to act onsubstrates IPP and (all-E)-FPP. The ellipses show the positions of coldauthentic samples, which are geraniol, farnesyl, and geranilgeranil fora, b and c respectively.

FIG. 3 is a photograph that indicates the autoradiograph of a thin layerchromatography which shows the products in the case of allowing themutant enzyme of the present invention produced in yeast to act onsubstrates IPP and (all-E)-GGPP. The ellipses show the positions of coldauthentic samples, which are geraniol, farnesyl, and geranilgeranil fora, b and c respectively.

FIG. 4 is a photograph that indicates the autoradiograph of a thin layerchromatography which shows the products in the case of allowing themutant enzyme of the present invention produced in E. coli to act on (A)substrates IPP and DMAPP, and on (B) substrates IPP and GPP. Theellipses show the positions of cold authentic samples, which aregeraniol, farnesyl, and geranilgeranil for a, b and c respectively.

FIG. 5 is the autoradiograph of a photograph that indicates a thin layerchromatography which shows the products in the case of allowing themutant enzyme of the present invention produced in E. coli to act on (A)substrates IPP and (all-E)-FPP, and on (B) substrates IPP and(all-E)-GGPP. The ellipses show the positions of cold authentic samples,which are geraniol, farnesyl, and geranilgeranil for a, b and crespectively.

DETAILED DESCRIPTION

As a specific example in the present invention, a geranylgeranyldiphosphate synthase (GGPS) gene of the hyper thermophilicarchaebacterium, Sulfolobus acidocaldarius, is used for the startingmaterial. The cloning method of this gene is described in detail in thespecification of Japanese Patent Application No. 6-315572. In addition,another example for cloning the gene is described in the presentspecification as Example 1, and a nucleotide sequence and an amino acidsequence encoded thereby are shown as SEQ ID NO: 1.

In the present invention, a cloned DNA is mutated in vitro. Althoughchemical treatment using a mutagen, or physical treatment using UV lightor X-rays can be used for the mutation means, chemical treatment isconvenient to carry out. Any routinely used chemical mutagen can be usedfor the mutagenesis for the present invention, an example of which isnitrite.

A specific example of mutagenesis is shown in Example 2.

The mutagenized DNA is inserted into a yeast expression vector toprepare a DNA library. Any vector that is able to express an insertedextraneous gene in the yeast can be used as an expression vector,examples of which include a yeast plasmid such as pYEUra3 (availablefrom Clonetech) and pYES2 (available from Invitrogen).

The resulting plasmid library is introduced into a yeast mutant straindefective for the ability to synthesize hexaprenyl diphosphate, which isone of the precursors of coenzyme Q6. Since this mutant strain is unableto synthesize coenzyme Q6 necessary for non-fermentative sugarmetabolism, it cannot be grown in medium that contains glycerol as thesole carbon source. Thus, if the yeast transformed by theabove-mentioned library is cultured in glycerol medium and the strainsthat grow are selected, strains can be selected that have acquired theability to synthesize prenyl diphosphate having a large number of carbonatoms for coenzyme Q synthesis.

Five positive clones were obtained in this manner from approximately1400 transformants. As a result of purifying the plasmids from theseclones, determining the nucleotide sequence of the inserted fragment,and predicting amino acid sequences that are coded, each mutant hadchanges in the amino acid sequence as indicated below.

Mutant 1: Methionine at position 85 changed to isoleucine, arginine atposition 199 changed to lysine, aspartic acid at position 312 changed toAsn

Mutant 2: Phenylalanine at position 118 changed to leucine

Mutant 3: Phenylalanine at position 77 changed to serine

Mutant 4: Phenylanine at position 77 changed to leucine and valine atposition 99 changed to methionine

Mutant 5: Phenylalanine at position 77 changed to serine and tyrosine atposition 101 changed to histidine

In contrast to wild-type enzymes being unable to synthesize prenyldiphosphate having at least 25 carbon atoms, enzymes having amino acidsequences containing these changes were able to synthesize prenyldiphosphate having 25 or more carbon atoms. Those amino acid sequenceshaving the above-mentioned amino acid substitutions are shown in SEQ IDNOs: 2 to 6.

Thus, it can be logically surmised that if an amino acid at any one ofthe above-mentioned positions is replaced with another amino acid, aprenyl diphosphate having more carbon atoms than that synthesized by thenative enzyme can be synthesized. Thus, the present invention provides amutant enzyme in which at least one amino acid from among phenylalanineat position 77, methionine at position 85, valine at position 99,tyrosine at position 101, phenylalanine at position 118, arginine atposition 199 and aspartic acid at position 312 is replaced with anotheramino acid, and said enzyme is able to synthesize prenyl diphosphatehaving at least 25 carbon atoms.

Particularly in the case that phenylalanine at position 77 is replacedwith another amino acid, and preferably a non-aromatic amino acid suchas serine or leucine, that enzyme is able to synthesize prenyldiphosphate having at least 25 carbon atoms. Thus, in one embodiment,the present invention provides an enzyme in which at least phenylalanineat position 77 is replaced with another amino acid such as serine,leucine or another non-aromatic amino acid. This type of enzyme includesenzymes in which replaced amino acids are present at one or a pluralityof the other above-mentioned positions. Examples of other amino acidpositions include valine at position 99 and/or tyrosine at position 101.

Thus, the present invention includes enzymes in which only phenylalanineat position 77 is replaced, enzymes in which phenylalanine at position77 and valine at position 99 are replaced, enzymes in whichphenylalanine at position 77 and tyrosine at position 101 are replaced,enzymes in which phenylalanine at position 77, valine at position 99 andtyrosine at position 101 are replaced, and enzymes in whichphenylalanine at position 77 and one or a plurality of amino acids atthe above-mentioned positions are replaced.

According to another mode of the present invention, an enzyme in whichmethionine at position 85, arginine at position 199 and aspartic acid atposition 312 are replaced with other amino acids is also able tosynthesize prenyl diphosphate having at least 25 carbon atoms. Thus, thepresent invention, in another embodiment, includes an enzyme in which atleast methionine at position 85, arginine at position 199 and asparticacid at position 312 are replaced with other amino acids. In thisembodiment, enzymes in which methionine at position 85, arginine atposition 199 and aspartic acid at position 312 are replaced, as well asenzymes containing amino acid replacements at one or a plurality ofsites other than at these sites or the above-mentioned mutation sites,are included.

According to still another embodiment of the present invention, anenzyme in which phenylalanine at position 118 is replaced with anotheramino acid can also synthesize prenyl diphosphate having at least 25carbon atoms. Thus, in another embodiment, the present inventionincludes enzymes in which at least the amino acid at position 119 isreplaced with another amino acid. In this embodiment, enzymes in whichthe amino acid at position 118 is replaced with another amino acid, aswell as enzymes containing amino acid replacements at one or a pluralityof positions of the above-mentioned amino acid replacement positions,are included.

Enzymes are known to have those own specificities of enzyme activitieseven in the case of being modified by addition, removal and/orreplacement of one or a few amino acids. Thus, in addition to thepeptides having the amino acid sequences shown in SEQ ID NOs: 2 to 6,the present invention also includes enzymes that the same specificitywhile having an amino acid sequence that is changed by replacing,deleting and/or adding one or a few, such as up to 5 or up to 10, aminoacids with respect to the amino acid sequences shown in SEQ ID Nos: 2 to6.

Two aspartate-rich domains (sites indicated with arrows in FIG. 1) areconserved in various prenyl transferases, and the diphosphate site ofthe substrate is thought to bind to these sites. Phenylalanine atposition 77 exists at the 5th position upstream to the N-terminal sidefrom the N-terminal of aspartate-rich domain I present on the N-terminalside among these two aspartate-rich domains. This phenylalanine isreplaced with a non-aromatic amino acid in 3 of the 5 mutants of thepresent invention.

Thus, in order to synthesize prenyl diphosphate having a large number ofcarbon atoms, for example that having 25 or more carbon atoms, ifphenylalanine at about the fifth position upstream to the N-terminalside from the amino acid of the N-terminal of aspartate-rich domain I isreplaced with another amino acid, for example a non-aromatic amino acid,even in the case of a prenyl transferase other than the prenyltransferase derived from Sulfolobus acidocaldarius having the amino acidsequence indicated in Sequence No. 1, an enzyme is obtained that is ableto synthesize prenyl diphosphate having a larger number of carbon atomsthan the wild type enzyme.

Thus, the present invention provides a process for producing a mutantprenyl transferase characterized by replacing phenylalanine at the 5thposition upstream to the N-terminal side from the amino acid of theN-terminal of aspartate-rich domain I of prenyl transferase. This aminoacid replacement can be performed by changing the codon that codes forthat amino acid.

In addition, the present invention provides a gene coding for thevarious above-mentioned mutant enzymes, a vector comprising that gene,particularly an expression vector, and a host transformed with saidvector. The gene (DNA) of the present invention can be easily obtainedby introducing a mutation into DNA that codes for the wild type aminoacid sequence indicated in SEQ ID NO: 1, according to routine methodssuch as site-directed mutagenesis or PCR.

Moreover, once the amino acid sequence of the target enzyme has beendetermined, a suitable nucleotide sequence that codes for it can bedetermined, thus making the mutant is possible to chemically synthesizeDNA by conventional DNA synthesis methods.

In addition, the present invention provides an expression vectorcomprising the DNA as described above, hosts transformed with saidexpression vector, and a process for producing an enzyme or peptide ofthe present invention using these hosts.

Although expression vectors contain an origin of replication, expressioncontrol sequence and so forth, these vary according to the host.Examples of hosts include procaryotes, examples of which includebacteria such as E. coli and Bacillus sp. including Bacillus subtilus;eucaryotes, examples,of which include yeasts such as Saccharomyces sp.including S. cerevisiae, and Pichia sp. including Pichia pastoris;molds, examples of which include Aspergillus sp. such as A. oryzae andA. niger; animal cells, examples of which include cultured cells andcultured cells of higher animals, such as CHO cells. In addition, it isalso possible to use plants for the host.

According to the present invention, as indicated in Examples,geranylfarnesyl diphosphate can be accumulated in the culture byculturing a host transformed by the DNA of the present invention, andgeranylfarnesyl diphosphate can be produced by recovering it from theculture. Also according to the present invention, geranylfarnesyldiphosphate can be produced by allowing the mutant GGPP synthaseproduced according to the process of the present invention to act on theisopentenyl diphosphate substrate and each allylic substrate such asfarnesyl diphosphate.

In an example of using E. coli for the host, gene regulation of geneexpression is known to exist such as in the process of transcribing mRNAfrom DNA and the process of translating protein from mRNA. In additionto those sequences present in nature (e.g. lac, trp, bla, lpp, P_(L),P_(R), ter, T3 and T7 as promoters), sequences in which their mutants(e.g. lacUV5) are artificially joined with wild type promoter sequences(e.g. tac, trc) are known as examples of promoter sequences thatregulate mRNA transcription, and these can also be used in the presentinvention.

It is known that the ribosome binding site (GAGG and other similarsequences) sequence and the distance to the initiation codon areimportant as sequences that regulate the activity to translate the MRNAto synthesize proteins. In addition, it is also well known that theterminator, which commands termination of transcription on the 3'-end(e.g. a vector containing rrnPT₁ T₂ is commercially available fromPharmacia), has an effect on protein synthesis efficiency in therecombinant.

Although commercially available products can be used as is for thevector that can be used for preparation of the recombinant vector of thepresent invention, various types of vectors induced according to aspecific purpose can also be used. Examples of these include pBR322,pBR327, pKK223-3, pRK233-2 and pTrc99, originating in pMB1 and havingthe replicon, pUC18, pUC19, pUC118, pUC119, pBluescript, pHSG298 andpHSG396, modified to improve the number of copies, pACYC177 andpACYC184, derived from p15A and having the replicon, as well as plasmidsoriginating in pSC101, ColE1, R1 and F factor. Moreover, expressionvectors, for fused proteins, that are easier to purify, can also beused, examples of which include pGEx-2T, pGEX-3X and pMal-c2, and theexample of a gene used as the starting material in the present inventionis described in Japanese Patent Application No. 6-315572,

In addition, gene introduction can also be performed by using virusvectors and transposons such as λ-phages and M13 phages in addition toplasmids. In the case of gene introduction into a microorganism otherthan E. coli, gene introduction into Bacillus sp. is known using pUB110(sold by Sigma) or pHY300PLK (sold by Takara Shuzo). These vectors aredescribed in Molecular Cloning (J. Sambrook, E. F. Fritsch, T. Maniatised., Cold Spring Harbor Laboratory Press, pub.), Cloning Vector (P. H.Pouwels, B. E. Enger Valk, W. J. Brammar ed., Elsevier pub.) and variouscompany catalogs.

Insertion of a DNA fragment coding for GGPP synthase and, as necessary,a DNA fragment having the function of regulating expression of the geneof the above-mentioned enzyme, into these vectors can be performedaccording to known methods using suitable restriction enzyme and ligase.Specific examples of plasmids of the invention prepared in this mannerinclude pBS-GGPSmut1, PBS-GGPSmut2, pBS-GGPSmut3, pBS-GGPSmut4 andpBS-GGPSmut5.

Examples of microorganisms that can be used for gene introduction withthis type of recombinant vector include

E. coli and Bacillus sp. This transformation can also be performedaccording to routine methods such as the CaCl₂ method or protoplastmethod described in Molecular Cloning (J. Sambrook, E. F. Fritsch, T.Maniatis ed., Cold Spring Harbor Laboratory Press pub.) and DNA CloningVol. I-III (D. M. Glover ed., IRL Press pub.).

In producing the mutant enzyme of the present invention, theabove-mentioned transformed cell is cultured after which the mutantenzyme can be collected and purified from that culture in accordancewith routine methods, examples of which include salting out, organicsolvent sedimentation, gel filtration, affinity chromatography,hydrophobic inter action chromatography and ion exchange chromatography.

In addition, the present invention provides a process for producingprenyl diphosphate using the enzyme of the present invention. In thisprocess, the enzyme of the present invention should be allowed to reactin a medium, and particularly an aqueous medium, and then the targetprenyl diphosphate should be recovered from the reaction medium asdesired. The enzyme may not only be purified enzyme, but also crudeenzymes obtained by semi-purification through various stages, or asubstance containing enzymes such as cultured microorganisms or theculture itself. In addition, the above-mentioned enzyme, crude enzyme orenzyme-containing substance may be an immobilized enzyme that has beenimmobilized in accordance with conventional methods.

Prenyl diphosphate having fewer carbon atoms than the target prenyldiphosphate, such as 5-20 carbon atoms and preferably less than 5 carbonatoms, and isopentyl diphosphate are used for the substrate. Water or anaqueous buffer, such as phosphate buffer, are used for the reactionmedium.

EXAMPLES

The following Examples provide a more detailed explanation of thepresent invention. Furthermore, the materials used in the followingExamples can all be easily acquired by a person with ordinary skill inthe art as described below.

Strain C296-LH3 of the budding yeast, Saccharomyces cerevisiae(Tzagoioff, A. and Dieckmann, C. L. (1990) Microbiological Reviews 54,211-255, Tzagoloff, A. et al. (1075) J. Bacteriol. 122, 826-831), wasused for the screening host.

Plasmid pG3/T1 (Tzagoloff, A. and Dieckmann, C. L. (1990)Microbiological Reviews 54, 211-255, Tzagoloff A. et al. (1975) J.Bacteriol. 122, 826-831, Ashby, M. N. and Edwards, P. A. (1990) J. Biol.Chem. 265, 13157-13164) or plasmid YEpG3ΔSpH, from which portions otherthan the HexPS coding region had been removed from pG3/T1 (Ashby, M. N.and Edwards, P. A. (1990) J. Biol. Chem. 265, 13157-13164), was used forthe positive control plasmid containing the HexPS gene.

Y-PGK, wherein the crtE gene portion had been removed from Y-crtE(Misawa, N. et al. (1990) J. Bacteriology 172, 6704-6712), was used forthe expression vector for library preparation. Saccharomyces cerevisiaestrain A451 was used as a wild strain used for the positive control.

However, the experimental materials required for the present inventionare not limited to those described above, but rather completely similarsubstitutes can also be used.

Screening host mutant strain C2960-LH3 for screening is a deficientstrain for the HexPS gene. In other words, a budding yeast HPS genefragment can easily be obtained from a widely known wild strain ofbudding yeast by PCR using an already known budding yeast HexPS genesequence (GenBank™/EMBL Data Bank accession number(s) JO5547). If thisgene fragment is then used by coupling with a yeast incorporatingplasmid (Y1p) such as pRS403, pRS404, pRS405 or pRS406 (available fromStratagene), an HexPS-deficient strain can easily be created by widelyconducted gene destruction using homologous recombination.

In addition, it also sufficient for the positive control plasmid if thisgene fragment is inserted using a widely known budding yeast expressionvector such as pYEUra3 (available from Clonetech) and pYES2 (availablefrom Invitrogen). The strain used for the positive control is notlimited to strain A451, but rather any strain is sufficient provided itretains the wild HexPS gene. In addition, it is sufficient to use acommercially available vector for the expression vector for librarypreparation such as pYEYra3 available from Clonetech or pYES2 availablefrom Invitrogen.

LKC-18 reversed phase thin layer chromatography plates were purchasedfrom Whatman Chemical Separation, Inc. 1-¹⁴ C!IPP was purchased fromAmersham.

EXAMPLE 1

Plasmid Construction

New HindIII restriction enzyme sites were introduced both upstream anddownstream of the GGPS gene (GenBank™/EMBL Data Bank accession numberD28748) of Sulfolobus acidocaldarius by PCR using the chemicallysynthesized DNA primers 5'-AAGAGAAGCTTATGAGTTACTTTGAC-3' (SEQ ID NO: 7)and 5'-GATACAAGCTTTATTTTCTCC-3' (SEQ ID NO: 8). Genomic DNA was purifiedin accordance with routine methods from Sulfolobus acidocaldarius,obtainable as ATCC33909 from the American Type Culture Collection(ATCC), and its clone DNA was then used for the template DNA of PCR.

The DNA fragment amplified with PCR was ligated to the HindIII site ofplasmid pBluescript (KS⁺) cleaved with HindIII to form pBS-GGPS. A crtEgene portion was removed by cleaving plasmid Y-crtE with HindIII, andthe remaining portion containing the PGK promoter and PGK terminator wasself-ligated to form Y-PGK. The insert portion containing GGPS geneobtained by severing pBS-GGPS with HindIII was introduced at the HindIIIsite of Y-PGK to form Y-GGPS.

EXAMPLE 2

Random Mutagenesis of GGPS Gene

A random mutation was introduced into the region coding for GGPS geneusing nitrite according to the method of Myers et al. (Myers, R. M. etal. (1985) Science 229, 242-247). Single strand DNA was isolated from E.coli containing pBS-GGPS by infection with helper phage M13K07, and thiswas then treated for 60 minutes with 1M sodium nitrite. Next, thecomplementary strand was synthesized as primer using chemical synthesisDNA 5'-CCCCCCTCGAGGTCGACGGTATCGATAA-3' (SEQ ID NO: 9) corresponding tothe sequence of the T7 promoter portion. The GGPS gene portion was thenextracted with HindIII restriction enzyme, introduced at the HindIIIsite of Y-PGK, and transformed to E. coli strain XL1-Blue to prepare thelibrary.

EXAMPLE 3

Yeast Transformation and Screening

The budding yeast, Saccharomyces cerevisiae, was transformed by thespheroplast method according to the method of Ashby et al. (Ashby, M. N.and Edwards, P. A. (1990) J. Biol. Chem. 265, 13157-13164). Namely,HexPS-deficient strain C296-LH3 was transformed with the previouslydescribed plasmid library and cultured on leucine-deficient agar plate(leu⁻ plate) using the top agar method (3% bactoagar, 0.67% yeastnitrogen base, 0.05% yeast extract, 0.05% bacto peptone, 1.0M sorbitoland 2% glucose).

The transformant having the Leu phenotype was inoculated onto YEPG (1%yeast extract, 2% ethanol, 2% bacto peptone and 3% glycerol), D (1%yeast extract, 2% ethanol, 2% bacto peptone, 3% glycerol and 0.1%glucose) and YPD (1% yeast extract, 2% bacto peptone and 2% glucose)agar media followed by incubation for 3 days at 30° C. Clones wereselected from the C2960-LH3 transformants with plasmid containingmutated GGPS that grew on the YEPG agar plate, grew and formed colonieslarger than those of non-transformed C296-LR3 on the D plate.

This complemented phenotype is considered to indicate that the electrontransport chain is active during the respiration reaction, or in otherwords, that a active coenzyme Q was synthesized in the C296-LH3 cells.Five clones having this complemented phenotype were obtained from 1,400transformants. As a result of retesting the resulting five clones, notonly were they able to grow on YEPG agar plates, but they also possessedthe ability to form colonies that were clearly larger than those ofYEpG3ΔSpH/C296-LH3, having a plasmid that contains HexPS gene of yeastorigin, on D agar plates. The plasmid DNA of these five clones werepurified in accordance with routine methods.

These plasmids were named Y-GGPSmut1, Y-GGPSmut2, Y-GGPSmut3, Y-GGPSmut4and Y-GGPSmut5.

Furthermore, since yeast strain C296-LH3 is deficient in HexPS activity,it is unable to biosynthesize coenzyme Q6 which has a hexaprenol groupon its side chain. Since coenzyme Q6 is required for non-fermentativemetabolism, C296-LH3 forms colonies on media containing a small amountof glucose that are smaller than those of the wild strain, and does notgrow on media that only contains a non-fermentative substrate likeglycerol for the carbon source. Prior to screening for mutated activity,the effects of expression in wild type GGPS derived from Sulfolobusacidocaldarius were investigated.

On the D plates, strain Y-GGPS/C296-LH3, which is strain C296-LH5 havinga plasmid containing the wild type GGPS gene, was found to clearly formcolonies smaller than those of YEpG3ΔSpH/C296-LH3 although intermediateto YEpG3ΔSpH/C296-LH3, possessing a plasmid containing HexPS gene ofyeast origin, and C296-LH3, not possessing a plasmid. However,Y-GGPS/C296-LH3 was unable to grow on the YEPG plate. This screeningmethod was therefore confirmed to be useful.

EXAMPLE 4

Determination of DNA Nucleotide Sequence and its Analysis

The nucleotide sequences of DNA coding for the five mutant GGPScontained in the five types of purified plasmids were determined usingthe Perkin-Elmer Model 373A Fluorescent DNA Sequencer according to thedideoxy chain termination method. Analysis of the nucleotide sequenceswas performed using the genetic data analysis software, MacMollyTetra.

The amino acid substitution sites as deduced from the nucleotidesequence of each mutant GGPS are shown in FIG. 1. Replacement sites werefound at the nucleotide sequence level for all selected mutants. In thecase of Mutant 1 which is the Y-GGPSmut1 insertion fragment,replacements were found consisting of mutant methionine at position 85changing to isoleucine, mutant arginine at position 199 changing tolysine, and mutant aspartic acid at position 312 changing to asparagine.In the case of Mutant 2 which is the Y-GGPSmut2 insertion fragment, theonly replacement was mutant phenylalanine at position 118 changing toleucine. In the case of Mutant 3 which is the Y-GGPmut3 insertionfragment, mutant Phe at position 77 changed to serine, in the case ofMutant 4 which is the Y-GGPSmut4 insertion fragment, mutantphenylalanine at position 77 changed to leucine and mutant valine atposition 99 changed to methionine, and in the case of Mutant 5 which isthe Y-GGPSmut5 insertion fragment, mutant phenylalanine at position 77changed to serine and mutant tyrosine at position 101 changed tohistidine.

A high proportion of these mutations consist of an aromatic amino acidresidue being replaced with a non-aromatic amino acid residue.Phenyalanine at position 77 in particular has the most significanteffect on the chain elongation reaction. Phenylalanine at position 77 islocated at the five residues upstream from the N-terminal residue of anaspartate-rich domain I. There are two aspartate-rich domain motifs(DDXX(XX)D) that are conserved in prenyl transferase. The diphosphateportion of the substrates are believed to bind here. The amino acidresidue located at the fifth position upstream from the N-terminalresidue of this aspartate-rich domain, which was focused on for thefirst time in the present invention, is considered to be extremelyimportant in determining the chain length of the reaction product.

EXAMPLE 5

A crude extract was prepared from the five selected clones(Y-GGPSmut1/C296-LH3, Y-GGPSmut2/C296-LH3, Y-GGPSmut3/C296-LH3,YGGPSmut4/C296-LH3 and Y-GGPSmut5/C-296-LH3) according to the method ofItoh et al. (Itoh, N. et al. (1984) J. Biol. Chem. 259, 13923-13929).

Namely, the above-mentioned yeast was incubated for 4 days at 30° C.Approximately 400 μg of cells were collected by centrifugation andwashed once with 800 μl of buffer A (50 mM Tris HCl pH 7.5, 5 mM MgCl₂,50 mM dithiothreitol, 1M sorbitol). The cells were then suspended in 1.2mM buffer B (50 mM Tris HCl pH 7.5, 5 mM MgCl₂, 3 mM dithiothreitol, 1Msorbitol) followed by the addition of 0.8 mg of zymolyase and incubationfor 1 hour at 30° C.

The prepared spheroblasts were washed three times with buffer B andsuspended in 1 ml of buffer C (50 mM Tris HCl pH 7.0, 10 mM2-mercaptoethanol, 1 mM phenylmethanesulfonyl fluoride, 1 mM EDTA).Ultrasonic treatment was performed 10 times on the suspension in ice attwo minute intervals, performing treatment for 10 seconds at a time atmaximum output using a Branson Sonifier. The lysates were incubated for1 hour at 55° C., and after inactivating prenyl transferase(s) of thehost cells, the lysates were centrifuged for 10 minutes at 10,000×g. Theresulting supernatant was used as a mutant GGPS crude enzyme solutionand assay of prenyl transferase activity.

The results of performing an assay of prenyl transferase activity byLKC-18 thin layer chromatography using this mutant GGPS crude enzymeliquid prepared from yeast are shown in FIGS. 2 and 3.

After carrying out the enzyme reaction at 55° C., polyprenyl diphosphatewas extracted with 1-butanol after which the 1-butanol was evaporatedwith a nitrogen gas flow. The resulting polyprenyl diphosphate wastreated with acid phosphatase in accordance with the method of Fujii etal. (Fujii et al. (1982) Biochim. Biophys. Acta. 712, 716-718). Thehydrolysis product was extracted with pentane and after performing thinlayer chromatography using acetone/H₂ O (9:1) for the developingsolution, the distribution of radioactivity was analyzed with the FujiiFilm Model BAS2000 Bio-image Analyzer. The alcohols as the authenticstandards, on which thin layer chromatography was performedsimultaneously, followed by staining with iodine vapor (geranyol,farnesol, geranylgeraniol), were used to determine the developinglocations.

FIG. 2 shows the result of reacting using 1-¹⁴ C!IPP and (all-E)-FPP forthe substrates, while FIG. 3 shows the result of reacting using 1-¹⁴C!IPP and (all-E)-GGPP for the substrates. Spots a through c correspondto the authentic standard samples, a indicating geraniol, b indicating(all-E)-farnesol, and c indicating (all-E)-geranylgeranyol. Oriindicates the sample-stopping point, S.F. indicates the solvent front.

On the basis of these results, in the case of using GGPP for the allylicsubstrate, it was shown that each mutant GGPS is able to synthesizegeranylfarnesyl diphosphate (GFPP) that is one isoprene unit longer thanthe reaction product of the wild type enzyme. On the other hand, thewild type GGPS is unable to synthesize the reaction product same as orlonger than the chain length of GFPP at a level that allows detection.In the case of using FPP for the allylic substrate, the product ratio ofGGPP/GFPP indicated by the mutant GGPS was different from each other.

EXAMPLE 6

Preparation of Mutant GGPS from E. coli

In order to ensure that the analysis is performed more accurately, eachmutant GGPS was over expressed in E. coli strain of XL 1-Blue. Namely,each of the five plasmids Y-GGPSmut1, Y-GGPSmut2, Y-GGPSmut3, Y-GGPSmut4and Y-GGPSmut5 obtained in screening was digested with HindIII to obtainHindIII DNA fragments that code for the mutant GGPS. These HindIII DNAfragments were inserted at the HindIII site of the plasmid vectorpBluescript (KS(+)) to obtain pBS-GGPSmut1, pBS-GGPSmut2, pBS-GGPSmut3,pBS-GPSmut4 and pBS-GGPSmut5 respectively.

E. coli XL1-Blue was transformed with pBS-GGPSmut1, pBS-GGPSmut2,pBS-GGPSmut3, pBS-GGPSmut4 and pBS-GGPSmut5 and cultured according tothe method described in Molecular Cloning (Sambrook, J. et al. (1989)Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.). After collecting the bacterialcells, the bacterial cells were ultrasonically homogenized in 50 mM TrisHCl buffer containing 10 mM 2-mercaptoethanol and 1 mM EDTA. After heattreating the homogenate for 1 hour at 55° C., it was centrifuged for 10minutes at 100,000×g. The supernatant was then collected as the crudeenzyme solution which was assayed for PTase activity.

Assay was performed by analysis of product with LKC-18 thin layerchromatography and by determination of enzyme activity. For thin layerchromatography, DMAPP, GPP, (all-E)-FPP and (all-E)-GGPP were used forthe allylic substrates, and after reacting in the same manner as Example5, LKC thin layer chromatography was performed in the same manner asExample 5. Those results are shown in FIGS. 4 and 5.

FIG. 4(A) is the result of reacting 1-¹⁴ C!IPP with DMAPP for thesubstrate, and (B) is the result of reacting 1-¹⁴ C!IPP with GPP for thesubstrate. FIG. 5(C) is a result of reacting 1-¹⁴ C!IPP with (all-E)-FPPfor the substrate, while (D) is a result of reacting 1-¹⁴ C!IPP with(all-E)-GGPP for the substrate. Ellipses a through c show the positionsof the authentic standard samples, a indicating geraniol, b indicating(all-E)-farnesol and c indicating (all-E)-geranylgeraniol. Ori indicatesthe sample-spotting point, while S.F. indicates the solvent front.

The prenyl transferase activity was assaied as follows. 1 ml of assaymixture, containing 25 nmol of 1-¹⁴ C!IPP (37 GBq/mol), 25 nmol ofallylic substrate (DMAPP, GPP, (all-E)-FPP or (all-E)-GGPP), 5 μmol ofMgCl₂, 10 μmol of phosphate buffer (pH 5.8) and the enzyme solution, wasincubated for 60 minutes at 55° C.

The reaction was stopped by cooling rapidly on ice. After adding 3.5 mlof water-saturated 1-butanol to the chilled mixture and shakingvigorously, the 1-butanol layer was washed with NaCl-saturated water and¹⁴ C radioactivity was measured with a liquid scintillation counter. 1unit of enzyme activity was defined as the amount for which 1 nmol of1-¹⁴ C!IPP is incorporated into elongated prenyl diphosphate (polyprenyldiphosphate) that can be extracted with the 1-butanol layer. Thoseresults are shown in the Table.

                  TABLE    ______________________________________    Relative   Product Distribution           Activity                   GPP     FPP    GGPP  GFPP   FFPP    Substrate           (dpm)   %       %      %     %      %    ______________________________________    Mutant 1    DMAPP  31,800  23.2    8.77   29.6  38.0   0.45    GPP     5,260  nd*     38.8   30.9  30.4   0.02    FPP     4,340  nd*     nd*    65.1  35.0   nd*    GGPP     998   nd*     nd*    nd*   100    nd*    Mutant 2    DMAPP  15,800  1.44    0.66   89.0   8.86  nd*    GPP     7,050  nd*     20.3   74.9   4.89  nd*    FPP     6,080  nd*     nd*    89.5  10.5   nd*    GGPP     379   nd*     nd*    nd*   100    nd*    Mutant 3    DMAPP  24,900  3.40    27.4   16.6  51.6   0.92    GPP     9,890  nd*     64.7    9.37 24.5   1.44    FPP     7,280  nd*     nd*    30.4  69.6   nd*    GGPP    3,200  nd*     nd*    nd*   100    nd*    Mutant 4    DMAPP  16,700  4.93    4.07   73.2  17.8   nd*    GPP     7,460  nd*     38.4   51.3  10.3   nd*    FPP     5,650  nd*     nd*    85.9  14.1   nd*    GGPP     551   nd*     nd*    nd*   100    nd*    Mutant 5    DMAPP  23,600  27.1    18.6   12.8  40.4   1.12    GPP     9,070  nd*     59.3   13.0  26.1   1.56    FPP     8,960  nd*     nd*    32.0  68.0   nd*    GGPP    2,200  nd*     nd*    nd*   100    nd*    Wild type    DMAPP  13,600  5.61    0.43   94.0  nd*    nd*    GPP     6,640  nd*     17.2   82.8  nd*    nd*    FPP     4,650  nd*     nd*    100   nd*    nd*    GGPP   nd*     nd*     nd*    nd*   nd*    nd*    ______________________________________     nd: Not detected

Each mutant GGPS exhibited activity that synthesizes polyprenyldiphosphate having a longer chain length than GGPP. The wild type GGPSas well as each mutant GGPS reacted the best with DMAPP amongst the fourallylic substrates. In addition, relative activity when allylicsubstrates were used that had a shorter chain length than FPP exhibitedsimilar values. However, relative activity and product distribution whenGGPP was used for the allylic substrate were considerably different.

When DMAPP, GPP and FPP were used for the allylic substrates, Mutant 1,which is coded for by the insert DNA of plasmid pBS-GGPSmut1, yieldedthe major reaction products of GFPP and GGPP. In particular, when DMAPPwas used for the allylic substrate, only a slight amount of hexaprenyldiphosphate (HexPP) was detected as the reaction product. Although thedistribution of reaction products varied between each allylic substrate,the proportion of product produced in one cycle of the condensationreaction was large.

In the case of Mutant 2 coded for by the insert DNA of plasmidpBS-GGPSmut2, the major product was GGPP and the proportion of GFPP wasroughly 10%. HexPP was not detected.

Mutant 3, which is coded for by the insert DNA of plasmid pBS-GGPSmut3,and Mutant 5, which is coded for by the insert DNA of plasmidpBS-GGPSmut5, demonstrated similar characteristics. These mutantsexhibited strong GFPP synthesis activity, while also synthesizing asmall amount of HexPP.

Mutant 4, which is coded for by the insert DNA of plasmid pBS-GGPSmut4,yielded GGPP as the major product, while the proportion of GFPP wasroughly 15%. FPP was effectively synthesized when GPP was used for theallylic substrate.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 9    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH:993    (B) TYPE:Nucleic acid    (C) STRANDEDNESS:Double    (D) TOPOLOGY:Linear    (ii) MOLECULE TYPE:Genomic DNA    (vi) ORIGINAL SOURCE:    (A) ORGANISM:Sulfolobus acidocaldarius    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    ATGAGTTACTTTGACAACTATTTTAATGAGATTGTTAATTCTGTAAAC48    MetSerTyrPheAspAsnTyrPheAsnGluIleValAsnSerValAsn    51015    GACATTATTAAGAGCTATATATCTGGAGATGTTCCTAAACTATATGAA96    AspIleIleLysSerTyrIleSerGlyAspValProLysLeuTyrGlu    202530    GCCTCATATCATTTGTTTACATCTGGAGGTAAGAGGTTAAGACCATTA144    AlaSerTyrHisLeuPheThrSerGlyGlyLysArgLeuArgProLeu    354045    ATCTTAACTATATCATCAGATTTATTCGGAGGACAGAGAGAAAGAGCT192    IleLeuThrIleSerSerAspLeuPheGlyGlyGlnArgGluArgAla    505560    TATTATGCAGGTGCAGCTATTGAAGTTCTTCATACTTTTACGCTTGTG240    TyrTyrAlaGlyAlaAlaIleGluValLeuHisThrPheThrLeuVal    65707580    CATGATGATATTATGGATCAAGATAATATCAGAAGAGGGTTACCCACA288    HisAspAspIleMetAspGlnAspAsnIleArgArgGlyLeuProThr    859095    GTCCACGTGAAATACGGCTTACCCTTAGCAATATTAGCTGGGGATTTA336    ValHisValLysTyrGlyLeuProLeuAlaIleLeuAlaGlyAspLeu    100105110    CTACATGCAAAGGCTTTTCAGCTCTTAACCCAGGCTCTTAGAGGTTTG384    LeuHisAlaLysAlaPheGlnLeuLeuThrGlnAlaLeuArgGlyLeu    115120125    CCAAGTGAAACCATAATTAAGGCTTTCGATATTTTCACTCGTTCAATA432    ProSerGluThrIleIleLysAlaPheAspIlePheThrArgSerIle    130135140    ATAATTATATCCGAAGGACAGGCAGTAGATATGGAATTTGAGGACAGA480    IleIleIleSerGluGlyGlnAlaValAspMetGluPheGluAspArg    145150155160    ATTGATATAAAGGAGCAGGAATACCTTGACATGATCTCACGTAAGACA528    IleAspIleLysGluGlnGluTyrLeuAspMetIleSerArgLysThr    165170175    GCTGCATTATTCTCGGCATCCTCAAGTATAGGCGCACTTATTGCTGGT576    AlaAlaLeuPheSerAlaSerSerSerIleGlyAlaLeuIleAlaGly    180185190    GCTAATGATAATGATGTAAGACTGATGTCTGATTTCGGTACGAATCTA624    AlaAsnAspAsnAspValArgLeuMetSerAspPheGlyThrAsnLeu    195200205    GGTATTGCATTTCAGATTGTTGACGATATCTTAGGTCTAACAGCAGAC672    GlyIleAlaPheGlnIleValAspAspIleLeuGlyLeuThrAlaAsp    210215220    GAAAAGGAACTTGGAAAGCCTGTTTTTAGTGATATTAGGGAGGGTAAA720    GluLysGluLeuGlyLysProValPheSerAspIleArgGluGlyLys    225230235240    AAGACTATACTTGTAATAAAAACACTGGAGCTTTGTAAAGAGGACGAG768    LysThrIleLeuValIleLysThrLeuGluLeuCysLysGluAspGlu    245250255    AAGAAGATTGTCCTAAAGGCGTTAGGTAATAAGTCAGCCTCAAAAGAA816    LysLysIleValLeuLysAlaLeuGlyAsnLysSerAlaSerLysGlu    260265270    GAATTAATGAGCTCAGCAGATATAATTAAGAAATACTCTTTAGATTAT864    GluLeuMetSerSerAlaAspIleIleLysLysTyrSerLeuAspTyr    275280285    GCATACAATTTAGCAGAGAAATATTATAAAAATGCTATAGACTCTTTA912    AlaTyrAsnLeuAlaGluLysTyrTyrLysAsnAlaIleAspSerLeu    290295300    AATCAAGTCTCCTCTAAGAGTGATATACCTGGAAAGGCTTTAAAATAT960    AsnGlnValSerSerLysSerAspIleProGlyLysAlaLeuLysTyr    305310315320    CTAGCTGAATTTACGATAAGAAGGAGAAAATAA993    LeuAlaGluPheThrIleArgArgArgLys    325330    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH:993    (B) TYPE:Nucleic acid    (C) STRANDEDNESS:Double    (D) TOPOLOGY:Linear    (ii) MOLECULE TYPE:Mutated genomic DNA    (xi) SEQUENCE DESCRIPTION:SEQ ID NO:2:    ATGAGTTACTTTGACAACTATTTTAATGAGATTGTTAATTCTGTAAAC48    MetSerTyrPheAspAsnTyrPheAsnGluIleValAsnSerValAsn    51015    GACATTATTAAGAGCTATATATCTGGAGATGTTCCTAAACTATATGAA96    AspIleIleLysSerTyrIleSerGlyAspValProLysLeuTyrGlu    202530    GCCTCATATCATTTGTTTACATCTGGAGGTAAGAGGTTAAGACCATTA144    AlaSerTyrHisLeuPheThrSerGlyGlyLysArgLeuArgProLeu    354045    ATCTTAACTATATCATCAGATTTATTCGGAGGACAGAGAGAAAGAGCT192    IleLeuThrIleSerSerAspLeuPheGlyGlyGlnArgGluArgAla    505560    TATTATGCAGGTGCAGCTATTGAAGTTCTTCATACTTTTACGCTTGTG240    TyrTyrAlaGlyAlaAlaIleGluValLeuHisThrPheThrLeuVal    65707580    CATGATGATATTATAGATCAAGATAATATCAGAAGAGGGTTACCCACA288    HisAspAspIleIleAspGlnAspAsnIleArgArgGlyLeuProThr    859095    GTCCACGTGAAATACGGCTTACCCTTAGCAATATTAGCTGGGGATTTA336    ValHisValLysTyrGlyLeuProLeuAlaIleLeuAlaGlyAspLeu    100105110    CTACATGCAAAGGCTTTTCAGCTCTTAACCCAGGCTCTTAGAGGTTTG384    LeuHisAlaLysAlaPheGlnLeuLeuThrGlnAlaLeuArgGlyLeu    115120125    CCAAGTGAAACCATAATTAAGGCTTTCGATATTTTCACTCGTTCAATA432    ProSerGluThrIleIleLysAlaPheAspIlePheThrArgSerIle    130135140    ATAATTATATCCGAAGGACAGGCAGTAGATATGGAATTTGAGGACAGA480    IleIleIleSerGluGlyGlnAlaValAspMetGluPheGluAspArg    145150155160    ATTGATATAAAGGAGCAGGAATACCTTGACATGATCTCACGTAAGACA528    IleAspIleLysGluGlnGluTyrLeuAspMetIleSerArgLysThr    165170175    GCTGCATTATTCTCGGCATCCTCAAGTATAGGCGCACTTATTGCTGGT576    AlaAlaLeuPheSerAlaSerSerSerIleGlyAlaLeuIleAlaGly    180185190    GCTAATGATAATGATGTAAAACTGATGTCTGATTTCGGTACGAATCTA624    AlaAsnAspAsnAspValLysLeuMetSerAspPheGlyThrAsnLeu    195200205    GGTATTGCATTTCAGATTGTTGACGATATCTTAGGTCTAACAGCAGAC672    GlyIleAlaPheGlnIleValAspAspIleLeuGlyLeuThrAlaAsp    210215220    GAAAAGGAACTTGGAAAGCCTGTTTTTAGTGATATTAGGGAGGGTAAA720    GluLysGluLeuGlyLysProValPheSerAspIleArgGluGlyLys    225230235240    AAGACTATACTTGTAATAAAAACACTGGAGCTTTGTAAAGAGGACGAG768    LysThrIleLeuValIleLysThrLeuGluLeuCysLysGluAspGlu    245250255    AAGAAGATTGTCCTAAAGGCGTTAGGTAATAAGTCAGCCTCAAAAGAA816    LysLysIleValLeuLysAlaLeuGlyAsnLysSerAlaSerLysGlu    260265270    GAATTAATGAGCTCAGCAGATATAATTAAGAAATACTCTTTAGATTAT864    GluLeuMetSerSerAlaAspIleIleLysLysTyrSerLeuAspTyr    275280285    GCATACAATTTAGCAGAGAAATATTATAAAAATGCTATAGACTCTTTA912    AlaTyrAsnLeuAlaGluLysTyrTyrLysAsnAlaIleAspSerLeu    290295300    AATCAAGTCTCCTCTAAGAGTAATATACCTGGAAAGGCTTTAAAATAT960    AsnGlnValSerSerLysSerAsnIleProGlyLysAlaLeuLysTyr    305310315320    CTAGCTGAATTTACGATAAGAAGGAGAAAATAA993    LeuAlaGluPheThrIleArgArgArgLys    325330    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH:993    (B) TYPE:Nucleic acid    (C) STRANDEDNESS:Double strand    (D) TOPOLOGY:Linear    (ii) MOLECULE TYPE: Mutated genomic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    ATGAGTTACTTTGACAACTATTTTAATGAGATTGTTAATTCTGTAAAC48    MetSerTyrPheAspAsnTyrPheAsnGluIleValAsnSerValAsn    51015    GACATTATTAAGAGCTATATATCTGGAGATGTTCCTAAACTATATGAA96    AspIleIleLysSerTyrIleSerGlyAspValProLysLeuTyrGlu    202530    GCCTCATATCATTTGTTTACATCTGGAGGTAAGAGGTTAAGACCATTA144    AlaSerTyrHisLeuPheThrSerGlyGlyLysArgLeuArgProLeu    354045    ATCTTAACTATATCATCAGATTTATTCGGAGGACAGAGAGAAAGAGCT192    IleLeuThrIleSerSerAspLeuPheGlyGlyGlnArgGluArgAla    505560    TATTATGCAGGTGCAGCTATTGAAGTTCTTCATACTTTTACGCTTGTG240    TyrTyrAlaGlyAlaAlaIleGluValLeuHisThrPheThrLeuVal    65707580    CATGATGATATTATGGATCAAGATAATATCAGAAGAGGGTTACCCACA288    HisAspAspIleMetAspGlnAspAsnIleArgArgGlyLeuProThr    859095    GTCCACGTGAAATACGGCTTACCCTTAGCAATATTAGCTGGGGATTTA336    ValHisValLysTyrGlyLeuProLeuAlaIleLeuAlaGlyAspLeu    100105110    CTACATGCAAAGGCTCTTCAGCTCTTAACCCAGGCTCTTAGAGGTTTG384    LeuHisAlaLysAlaLeuGlnLeuLeuThrGlnAlaLeuArgGlyLeu    115120125    CCAAGTGAAACCATAATTAAGGCTTTCGATATTTTCACTCGTTCAATA432    ProSerGluThrIleIleLysAlaPheAspIlePheThrArgSerIle    130135140    ATAATTATATCCGAAGGACAGGCAGTAGATATGGAATTTGAGGACAGA480    IleIleIleSerGluGlyGlnAlaValAspMetGluPheGluAspArg    145150155160    ATTGATATAAAGGAGCAGGAATACCTTGACATGATCTCACGTAAGACA528    IleAspIleLysGluGlnGluTyrLeuAspMetIleSerArgLysThr    165170175    GCTGCATTATTCTCGGCATCCTCAAGTATAGGCGCACTTATTGCTGGT576    AlaAlaLeuPheSerAlaSerSerSerIleGlyAlaLeuIleAlaGly    180185190    GCTAATGATAATGATGTAAGACTGATGTCTGATTTCGGTACGAATCTA624    AlaAsnAspAsnAspValArgLeuMetSerAspPheGlyThrAsnLeu    195200205    GGTATTGCATTTCAGATTGTTGACGATATCTTAGGTCTAACAGCAGAC672    GlyIleAlaPheGlnIleValAspAspIleLeuGlyLeuThrAlaAsp    210215220    GAAAAGGAACTTGGAAAGCCTGTTTTTAGTGATATTAGGGAGGGTAAA720    GluLysGluLeuGlyLysProValPheSerAspIleArgGluGlyLys    225230235240    AAGACTATACTTGTAATAAAAACACTGGAGCTTTGTAAAGAGGACGAG768    LysThrIleLeuValIleLysThrLeuGluLeuCysLysGluAspGlu    245250255    AAGAAGATTGTCCTAAAGGCGTTAGGTAATAAGTCAGCCTCAAAAGAA816    LysLysIleValLeuLysAlaLeuGlyAsnLysSerAlaSerLysGlu    260265270    GAATTAATGAGCTCAGCAGATATAATTAAGAAATACTCTTTAGATTAT864    GluLeuMetSerSerAlaAspIleIleLysLysTyrSerLeuAspTyr    275280285    GCATACAATTTAGCAGAGAAATATTATAAAAATGCTATAGACTCTTTA912    AlaTyrAsnLeuAlaGluLysTyrTyrLysAsnAlaIleAspSerLeu    290295300    AATCAAGTCTCCTCTAAGAGTGATATACCTGGAAAGGCTTTAAAATAT960    AsnGlnValSerSerLysSerAspIleProGlyLysAlaLeuLysTyr    305310315320    CTAGCTGAATTTACGATAAGAAGGAGAAAATAA993    LeuAlaGluPheThrIleArgArgArgLys    325330    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH:993    (B) TYPE:Nucleic acid    (C) STRANDEDNESS:Double strand    (D) TOPOLOGY:Linear    (ii) MOLECULE TYPE:Mutated genomic DNA    (xi) SEQUENCE DESCRIPTION:SEQ ID NO:4:    ATGAGTTACTTTGACAACTATTTTAATGAGATTGTTAATTCTGTAAAC48    MetSerTyrPheAspAsnTyrPheAsnGluIleValAsnSerValAsn    51015    GACATTATTAAGAGCTATATATCTGGAGATGTTCCTAAACTATATGAA96    AspIleIleLysSerTyrIleSerGlyAspValProLysLeuTyrGlu    202530    GCCTCATATCATTTGTTTACATCTGGAGGTAAGAGGTTAAGACCATTA144    AlaSerTyrHisLeuPheThrSerGlyGlyLysArgLeuArgProLeu    354045    ATCTTAACTATATCATCAGATTTATTCGGAGGACAGAGAGAAAGAGCT192    IleLeuThrIleSerSerAspLeuPheGlyGlyGlnArgGluArgAla    505560    TATTATGCAGGTGCAGCTATTGAAGTTCTTCATACTTCTACGCTTGTG240    TyrTyrAlaGlyAlaAlaIleGluValLeuHisThrSerThrLeuVal    65707580    CATGATGATATTATGGATCAAGATAATATCAGAAGAGGGTTACCCACA288    HisAspAspIleMetAspGlnAspAsnIleArgArgGlyLeuProThr    859095    GTCCACGTGAAATACGGCTTACCCTTAGCAATATTAGCTGGGGATTTA336    ValHisValLysTyrGlyLeuProLeuAlaIleLeuAlaGlyAspLeu    100105110    CTACATGCAAAGGCTTTTCAGCTCTTAACCCAGGCTCTTAGAGGTTTG384    LeuHisAlaLysAlaPheGlnLeuLeuThrGlnAlaLeuArgGlyLeu    115120125    CCAAGTGAAACCATAATTAAGGCTTTCGATATTTTCACTCGTTCAATA432    ProSerGluThrIleIleLysAlaPheAspIlePheThrArgSerIle    130135140    ATAATTATATCCGAAGGACAGGCAGTAGATATGGAATTTGAGGACAGA480    IleIleIleSerGluGlyGlnAlaValAspMetGluPheGluAspArg    145150155160    ATTGATATAAAGGAGCAGGAATACCTTGACATGATCTCACGTAAGACA528    IleAspIleLysGluGlnGluTyrLeuAspMetIleSerArgLysThr    165170175    GCTGCATTATTCTCGGCATCCTCAAGTATAGGCGCACTTATTGCTGGT576    AlaAlaLeuPheSerAlaSerSerSerIleGlyAlaLeuIleAlaGly    180185190    GCTAATGATAATGATGTAAGACTGATGTCTGATTTCGGTACGAATCTA624    AlaAsnAspAsnAspValArgLeuMetSerAspPheGlyThrAsnLeu    195200205    GGTATTGCATTTCAGATTGTTGACGATATCTTAGGTCTAACAGCAGAC672    GlyIleAlaPheGlnIleValAspAspIleLeuGlyLeuThrAlaAsp    210215220    GAAAAGGAACTTGGAAAGCCTGTTTTTAGTGATATTAGGGAGGGTAAA720    GluLysGluLeuGlyLysProValPheSerAspIleArgGluGlyLys    225230235240    AAGACTATACTTGTAATAAAAACACTGGAGCTTTGTAAAGAGGACGAG768    LysThrIleLeuValIleLysThrLeuGluLeuCysLysGluAspGlu    245250255    AAGAAGATTGTCCTAAAGGCGTTAGGTAATAAGTCAGCCTCAAAAGAA816    LysLysIleValLeuLysAlaLeuGlyAsnLysSerAlaSerLysGlu    260265270    GAATTAATGAGCTCAGCAGATATAATTAAGAAATACTCTTTAGATTAT864    GluLeuMetSerSerAlaAspIleIleLysLysTyrSerLeuAspTyr    275280285    GCATACAATTTAGCAGAGAAATATTATAAAAATGCTATAGACTCTTTA912    AlaTyrAsnLeuAlaGluLysTyrTyrLysAsnAlaIleAspSerLeu    290295300    AATCAAGTCTCCTCTAAGAGTGATATACCTGGAAAGGCTTTAAAATAT960    AsnGlnValSerSerLysSerAspIleProGlyLysAlaLeuLysTyr    305310315320    CTAGCTGAATTTACGATAAGAAGGAGAAAATAA993    LeuAlaGluPheThrIleArgArgArgLys    325330    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH:993    (B) TYPE:Nucleic acid    (C) STRANDEDNESS:Double strand    (D) TOPOLOGY:Linear    (ii) MOLECULE TYPE:Mutated genomic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    ATGAGTTACTTTGACAACTATTTTAATGAGATTGTTAATTCTGTAAAC48    MetSerTyrPheAspAsnTyrPheAsnGluIleValAsnSerValAsn    51015    GACATTATTAAGAGCTATATATCTGGAGATGTTCCTAAACTATATGAA96    AspIleIleLysSerTyrIleSerGlyAspValProLysLeuTyrGlu    202530    GCCTCATATCATTTGTTTACATCTGGAGGTAAGAGGTTAAGACCATTA144    AlaSerTyrHisLeuPheThrSerGlyGlyLysArgLeuArgProLeu    354045    ATCTTAACTATATCATCAGATTTATTCGGAGGACAGAGAGAAAGAGCT192    IleLeuThrIleSerSerAspLeuPheGlyGlyGlnArgGluArgAla    505560    TATTATGCAGGTGCAGCTATTGAAGTTCTTCATACTCTTACGCTTGTG240    TyrTyrAlaGlyAlaAlaIleGluValLeuHisThrLeuThrLeuVal    65707580    CATGATGATATTATGGATCAAGATAATATCAGAAGAGGGTTACCCACA288    HisAspAspIleMetAspGlnAspAsnIleArgArgGlyLeuProThr    859095    GTCCACATGAAATACGGCTTACCCTTAGCAATATTAGCTGGGGATTTA336    ValHisMetLysTyrGlyLeuProLeuAlaIleLeuAlaGlyAspLeu    100105110    CTACATGCAAAGGCTTTTCAGCTCTTAACCCAGGCTCTTAGAGGTTTG384    LeuHisAlaLysAlaPheGlnLeuLeuThrGlnAlaLeuArgGlyLeu    115120125    CCAAGTGAAACCATAATTAAGGCTTTCGATATTTTCACTCGTTCAATA432    ProSerGluThrIleIleLysAlaPheAspIlePheThrArgSerIle    130135140    ATAATTATATCCGAAGGACAGGCAGTAGATATGGAATTTGAGGACAGA480    IleIleIleSerGluGlyGlnAlaValAspMetGluPheGluAspArg    145150155160    ATTGATATAAAGGAGCAGGAATACCTTGACATGATCTCACGTAAGACA528    IleAspIleLysGluGlnGluTyrLeuAspMetIleSerArgLysThr    165170175    GCTGCATTATTCTCGGCATCCTCAAGTATAGGCGCACTTATTGCTGGT576    AlaAlaLeuPheSerAlaSerSerSerIleGlyAlaLeuIleAlaGly    180185190    GCTAATGATAATGATGTAAGACTGATGTCTGATTTCGGTACGAATCTA624    AlaAsnAspAsnAspValArgLeuMetSerAspPheGlyThrAsnLeu    195200205    GGTATTGCATTTCAGATTGTTGACGATATCTTAGGTCTAACAGCAGAC672    GlyIleAlaPheGlnIleValAspAspIleLeuGlyLeuThrAlaAsp    210215220    GAAAAGGAACTTGGAAAGCCTGTTTTTAGTGATATTAGGGAGGGTAAA720    GluLysGluLeuGlyLysProValPheSerAspIleArgGluGlyLys    225230235240    AAGACTATACTTGTAATAAAAACACTGGAGCTTTGTAAAGAGGACGAG768    LysThrIleLeuValIleLysThrLeuGluLeuCysLysGluAspGlu    245250255    AAGAAGATTGTCCTAAAGGCGTTAGGTAATAAGTCAGCCTCAAAAGAA816    LysLysIleValLeuLysAlaLeuGlyAsnLysSerAlaSerLysGlu    260265270    GAATTAATGAGCTCAGCAGATATAATTAAGAAATACTCTTTAGATTAT864    GluLeuMetSerSerAlaAspIleIleLysLysTyrSerLeuAspTyr    275280285    GCATACAATTTAGCAGAGAAATATTATAAAAATGCTATAGACTCTTTA912    AlaTyrAsnLeuAlaGluLysTyrTyrLysAsnAlaIleAspSerLeu    290295300    AATCAAGTCTCCTCTAAGAGTGATATACCTGGAAAGGCTTTAAAATAT960    AsnGlnValSerSerLysSerAspIleProGlyLysAlaLeuLysTyr    305310315320    CTAGCTGAATTTACGATAAGAAGGAGAAAATAA993    LeuAlaGluPheThrIleArgArgArgLys    325330    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH:993    (B) TYPE:Nucleic acid    (C) STRANDEDNESS:Double strand    (D) TOPOLOGY:Linear    (ii) MOLECULE TYPE:Mutated genomic DNA    (xi) SEQUENCE DESCRIPTION:SEQ ID NO:6:    ATGAGTTACTTTGACAACTATTTTAATGAGATTGTTAATTCTGTAAAC48    MetSerTyrPheAspAsnTyrPheAsnGluIleValAsnSerValAsn    51015    GACATTATTAAGAGCTATATATCTGGAGATGTTCCTAAACTATATGAA96    AspIleIleLysSerTyrIleSerGlyAspValProLysLeuTyrGlu    202530    GCCTCATATCATTTGTTTACATCTGGAGGTAAGAGGTTAAGACCATTA144    AlaSerTyrHisLeuPheThrSerGlyGlyLysArgLeuArgProLeu    354045    ATCTTAACTATATCATCAGATTTATTCGGAGGACAGAGAGAAAGAGCT192    IleLeuThrIleSerSerAspLeuPheGlyGlyGlnArgGluArgAla    505560    TATTATGCAGGTGCAGCTATTGAAGTTCTTCATACTTCTACGCTTGTG240    TyrTyrAlaGlyAlaAlaIleGluValLeuHisThrSerThrLeuVal    65707580    CATGATGATATTATGGATCAAGATAATATCAGAAGAGGGTTACCCACA288    HisAspAspIleMetAspGlnAspAsnIleArgArgGlyLeuProThr    859095    GTCCACGTGAAACACGGCTTACCCTTAGCAATATTAGCTGGGGATTTA336    ValHisValLysHisGlyLeuProLeuAlaIleLeuAlaGlyAspLeu    100105110    CTACATGCAAAGGCTTTTCAGCTCTTAACCCAGGCTCTTAGAGGTTTG384    LeuHisAlaLysAlaPheGlnLeuLeuThrGlnAlaLeuArgGlyLeu    115120125    CCAAGTGAAACCATAATTAAGGCTTTCGATATTTTCACTCGTTCAATA432    ProSerGluThrIleIleLysAlaPheAspIlePheThrArgSerIle    130135140    ATAATTATATCCGAAGGACAGGCAGTAGATATGGAATTTGAGGACAGA480    IleIleIleSerGluGlyGlnAlaValAspMetGluPheGluAspArg    145150155160    ATTGATATAAAGGAGCAGGAATACCTTGACATGATCTCACGTAAGACA528    IleAspIleLysGluGlnGluTyrLeuAspMetIleSerArgLysThr    165170175    GCTGCATTATTCTCGGCATCCTCAAGTATAGGCGCACTTATTGCTGGT576    AlaAlaLeuPheSerAlaSerSerSerIleGlyAlaLeuIleAlaGly    180185190    GCTAATGATAATGATGTAAGACTGATGTCTGATTTCGGTACGAATCTA624    AlaAsnAspAsnAspValArgLeuMetSerAspPheGlyThrAsnLeu    195200205    GGTATTGCATTTCAGATTGTTGACGATATCTTAGGTCTAACAGCAGAC672    GlyIleAlaPheGlnIleValAspAspIleLeuGlyLeuThrAlaAsp    210215220    GAAAAGGAACTTGGAAAGCCTGTTTTTAGTGATATTAGGGAGGGTAAA720    GluLysGluLeuGlyLysProValPheSerAspIleArgGluGlyLys    225230235240    AAGACTATACTTGTAATAAAAACACTGGAGCTTTGTAAAGAGGACGAG768    LysThrIleLeuValIleLysThrLeuGluLeuCysLysGluAspGlu    245250255    AAGAAGATTGTCCTAAAGGCGTTAGGTAATAAGTCAGCCTCAAAAGAA816    LysLysIleValLeuLysAlaLeuGlyAsnLysSerAlaSerLysGlu    260265270    GAATTAATGAGCTCAGCAGATATAATTAAGAAATACTCTTTAGATTAT864    GluLeuMetSerSerAlaAspIleIleLysLysTyrSerLeuAspTyr    275280285    GCATACAATTTAGCAGAGAAATATTATAAAAATGCTATAGACTCTTTA912    AlaTyrAsnLeuAlaGluLysTyrTyrLysAsnAlaIleAspSerLeu    290295300    AATCAAGTCTCCTCTAAGAGTGATATACCTGGAAAGGCTTTAAAATAT960    AsnGlnValSerSerLysSerAspIleProGlyLysAlaLeuLysTyr    305310315320    CTAGCTGAATTTACGATAAGAAGGAGAAAATAA993    LeuAlaGluPheThrIleArgArgArgLys    325330    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH:26    (B) TYPE: Nucleic acid    (C) STRANDEDNESS:Single strand    (D) TOPOLOGY:Linear    (ii) MOLECULE TYPE:Synthetic DNA    (xi) SEQUENCE DESCRIPTION:SEQ ID NO:7:    AAGAGAAGCTTATGAGTTACTTTGAC26    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH:21    (B) TYPE:Nucleic acid    (C) STRANDEDNESS:Single strand    (D) TOPOLOGY:Linear    (ii) MOLECULE TYPE:Synthetic DNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    GATACAAGCTTTATTTTCTCC21    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH:28    (B) TYPE:Nucleic acid    (C) STRANDEDNESS:Single strand    (D) TOPOLOGY:Linear    (ii) MOLECULE TYPE:Synthetic DNA    (xi) SEQUENCE DESCRIPTION:SEQ ID NO:9:    CCCCCCTCGAGGTCGACGGTATCGATAA28    __________________________________________________________________________

We claim:
 1. A process for production of prenyl diphosphate whichcomprises at least 25 carbon atoms, said process comprising reacting asubstrate with mutated Sulfolobus acidocaldarius geranylgeranyldiphosphate synthase having an amino acid sequence which differs fromthat of wild-type Sulfolobus acidocaldarius geranylgeranyl diphosphatesynthase encoded by SEQ ID NO: 1, wherein said mutated geranylgeranyldiphosphate synthase is selected from the group consisting ofMutant 1,which has isoleucine at amino acid position 85, lysine at amino acidposition 199, and asparagine at amino acid position 312; Mutant 2, whichhas leucine at amino acid position 118; Mutant 3, which has serine atamino acid position 77; Mutant 4, which has leucine at amino acidposition 77 and methionine at amino acid position 99; and Mutant 5,which has serine at amino acid position 77 and histidine at amino acidposition 101; said substrate selected from the group consisting ofisopentenyl diphosphate, dimethylallyl diphosphate, geranyl diphosphate,farnesyl diphosphate and geranylgeranyl diphosphate.
 2. A process forproduction of prenyl diphosphate which comprises at least 25 carbonatoms, said process comprising reacting a substrate with a recombinantenzyme synthesized by host cells which have been transformed with a geneencoding a mutant prenyl diphosphate synthase, said gene containing anucleotide codon encoding a non-aromatic amino acid residue located atposition five amino acids upstream of the amino terminal end of theaspartic acid-rich domain 1 of prenyl diphosphate synthase, wherein thewild-type enzyme is encoded by SEQ ID NO:1,said substrate selected fromthe group consisting of isopentenyl diphosphate, dimethylallyldiphosphate, geranyl diphosphate, farnesyl diphosphate andgeranylgeranyl diphosphate.